Process for production of methylcobalamin

ABSTRACT

The present invention provides an industrially excellent and novel process for producing methylcobalamin useful as medicines. Namely, it provides a process for producing methylcobalamin, which comprises the step of methylating cyanocobalamin or hydroxocobalamin in the presence of a reducing agent and a water-soluble methylating agent.

This application is the national phase under 35 U.S.C. §371 of PCTInternational Application No. PCT/JP00/08675 which has an Internationalfiling date of Dec. 7, 2000, which designated the United States ofAmerica.

FIELD OF THE INVENTION

Methylcobalamin is a coenzyme-type vitamin B₁₂ existing in blood andcerebrospinal fluid and is excellent in migrating ability to nervoustissues as compared with other B₁₂ homologs. Biochemically, it exhibitsa pharmacological action of accelerating metabolism of nucleic acids,proteins and lipids by methyl group rearrangement and thereby restoringdamaged nervous tissues. Based on these properties, it has beenclinically employed for preventing, treating or improving peripheralneuropathy such as diabetic neuropathy and polyneuritis, particularlynumbness, pain and paralysis, and is also effective in megaloblasticanemia owing to vitamin B₁₂ deficiency, and thus, it is an importantvitamin.

Accordingly, the present invention relates to an industrially excellentand novel process for producing methylcobalamin useful as medicines.

PRIOR ART

Methylcobalamin has been hitherto produced mainly by the followingsynthetic methods:

(1) a method of reacting hydroxocobalamin with a dicarboxylic acidmonomethyl ester in the presence of a metal powder (JP-A 49-47899);

(2) a method of reacting cyanocobalamin with monomethyl oxalate in thepresence of a metal powder in hydrous methanol (JP-A 50-41900);

(3) a method of reacting hydroxocobalamin with methylmercury iodide orammonium methylhexafluorosilicate (JP-B 50-38120); and

(4) a method of reacting cyanocobalamin with methyl iodide in thepresence of sodium borohydride (JP-B 45-38059).

However, dicarboxylic acid monomethyl esters such as monomethyl oxalateto be used in the methods (1) and (2) are not commercially available andhence are necessary to prepare in use, so that it is impossible toutilize them industrially. Furthermore, zinc powder to be used as themetal powder is a heavy metal and hence it is inevitable to takemeasures for preventing its contamination into products and forprotecting the environment, so that the powder is industrially notpreferable.

Moreover, methylmercury iodide to be used in (3) is a pollutant andhence cannot be employed industrially. Furthermore, ammoniummethylhexafluorosilicate is also not commercially available and hence isnecessary to prepare in use, so that it is impossible to utilize itindustrially.

On the other hand, the synthetic method (4) is a very excellent methodin view of yield and product purity, but is not satisfactory as anindustrial process because methyl iodide has an extremely low boilingpoint (41 to 43° C.) and is difficult to handle. Furthermore, from theviewpoint of protecting working environment or natural environment, theuse of methyl iodide assigned as a specified chemical substance andhaving toxicity such as possibility of carcinogenicity is by no meanspreferable in view of industrial health of factory workers. Moreover, inorder to obtain highly pure methylcobalamin by the method of usingmethyl iodide, operation for purification by one or more kinds of columnchromatography is usually necessary, which is a serious problem fromoperational viewpoint and viewpoint of production cost. In addition, thequantity of organic solvents for use in the column purification is largeand also waste liquid quantity tends to be enormous.

Thus, an industrially excellent process for producing methylcobalamin isnot completely established yet and hence a novel excellent method hasbeen desired.

DISCLOSURE OF THE INVENTION

The present inventors have extensively studied for the purpose ofimproving the above problems. As a result, surprisingly, they have foundthat aimed methylcobalamin can be conveniently, safely, andinexpensively obtained in high yields by the below-mentioned method, andthus accomplished the present invention.

Accordingly, the present invention provides an industrially excellentprocess for producing methylcobalamin, particularly a novel processusing no methyl iodide and no purification by column chromatography.

The following will explain the present invention in detail.

The present invention relates to a process for producing methylcobalamin(V), which is represented by the following chemical reaction formula:

Cobalamin-CN or Cobalamin-OH→Cobalamin-CH₃

Cyanocobalamin (I), hydroxocobalamin (II), and methylcobalamin (V)according to the present invention are known natural compounds and arerepresented by the following chemical formula:

Cyanocobalamin, CAS Res. No.: 68-19-9

Hydroxocobalamin, CAS Res. No.: 13422-51-0

Methylcobalamin, CAS Res. No.: 13422-55-4

R²═CN: Cyanocobalamin (I)

R²═OH: Hydroxocobalamin (II)

R²═CH₃: Methylcobalamin (V)

The characteristic feature of the present invention is that a highlypure methylcobalamin equal to or superior to the product purified bycolumn chromatography can be conveniently obtained in high yields onlyby methylating cyanocobalamin (I) or hydroxocobalamin (II) in thepresence of a reducing agent (III) and a water-soluble methylating agent(IV) usually in an aqueous solution or a hydrous organic solvent, ifnecessary, precipitating the reaction product which is hardly soluble inwater as crystals or precipitates, and then separating and treating it.

The water-soluble methylating agent (IV) in the present invention is notlimited as far as it's solubility in water is 2% or more, andspecifically includes trimethylsulfur derivatives (VI) represented bythe following formula, for example.

wherein X represents a halogen atom or methoxysulfonyloxy group; and nrepresents 0 or 1.

Examples of the trimethylsulfur derivatives (VI) include the followingcompounds but they are not limited thereto.

(1) Trimethylsulfoxonium iodide, CAS Res. No.: 1774-47-6

(2) Trimethylsulfonium iodide, CAS Res. No.: 2181-42-2

(3) Trimethylsulfoxonium chloride, CAS Res. No.: 5034-06-0

(4) Trimethylsulfonium chloride, CAS Res. No.: 3086-29-1

(5) Trimethylsulfoxonium bromide, CAS Res. No.: 3084-53-5

(6) Trimethylsulfoxonium bromide, CAS Res. No.: 25596-24-1

(7) Trimethylsulfonium methyl sulfate, CAS Res. No.: 2181-44-4

All these compounds are known products and, in particular,trimethylsulfoxonium iodide, trimethylsulfonium iodide,trimethylsulfoxonium chloride, trimethylsulfoxonium bromide andtrimethylsulfonium bromide are inexpensive and available as industrialstarting materials. Moreover, trimethylsulfonium chloride can be easilysynthesized and available by the method described in Tetrahedron Lett.,27, 1233 (1986) (B. Byrne et al.).

Among the trimethylsulfur derivatives (VI), trimethylsulfoxoniumbromide, trimethylsulfonium bromide, trimethylsulfoxonium chloride andtrimethylsulfonium chloride particularly exhibit a high solubility inwater and have a characteristic that the use in a smaller amount affordshighly pure methylcobalamin in high yields.

The amount of the trimethylsulfur derivative (VI) to be used is notparticularly limited, but it is used in an amount of usually 1.0 to 5equivalents, preferably 1.1 to 4.5 equivalents and more preferably 1.2to 4 equivalents to cyanocobalamin (I) or hydroxocobalamin (II).

The reducing agent (III) according to the present invention is notparticularly limited as far as it is a reducing agent employable in thesynthesis of cyanocobalamin (I) or hydroxocobalamin (II). Morespecifically, examples thereof include sodium borohydride.

The amount of the reducing agent (III) to be used is not particularlylimited, but it is used in an amount of usually 5 to 30 equivalents,preferably 8 to 25 equivalents and more preferably 10 to 20 equivalentsto cyanocobalamin (I) or hydroxocobalamin (II).

The process according to the present invention enables the production ofhighly pure methylcobalamin in high yields using no metal ion or usingonly a small amount thereof as a cyan ion-trapping agent, and theprocess exhibits an extremely excellent effect in view that no problemarises at removal of metal ion products, which is difficult to filter,from the system.

Generally, when and methyl iodide is used as a methylating agent,ferrous sulfate is used as a cyan ion-trapping agent in combination withthose agents in most cases, and it is necessary to use ferrous sulfatein an amount of at least 30% by weight or more relative tocyanocobalamin (I) or hydroxocobalamin (II).

However, in the present invention, it is possible to obtain highly puremethylcobalamin in high yields because methylation proceeds even when noferrous sulfate is used as a cyan ion-trapping agent.

Furthermore, in the case that ferrous sulfate is used in a small amountas a cyan ion-trapping agent, the reaction proceeds more rapidly andhighly pure methylcobalamin can be obtained in high yields by the samepost-treatment as in the case that no ferrous sulfate is used. Moreover,in the case that cobalt chloride is used in a small amount, highly puremethylcobalamin can be also obtained in high yields because themethylation proceeds highly selectively and hence the production ofimpurities is inhibited.

Therefore, the present invention also relates to a process for producingmethylcobalamin (V), which comprises the steps of methylatingcyanocobalamin (I) or hydroxocobalamin (II) in the presence of a cyanion-trapping agent, a reducing agent (III) and a water-solublemethylating agent (IV) in an aqueous solution or a hydrous organicsolvent; and then precipitating the reaction product as crystals orprecipitates.

In the present invention, in the case that a cyan ion-trapping agent isused, examples of the cyan ion-trapping agent include metals or metalsalts such as ferrous sulfate, iron powder, Mohr's salt, ferrouschloride, cobalt chloride, nickel chloride and zinc chloride, andparticularly preferred are ferrous sulfate and/or cobalt chloride. Thesemetals or metal salts may be used solely or in combination.

The cyan ion-trapping agent may be used in a small amount, and theamount is usually from 1 to 30% by weight and more preferably from 1 to10% by weight to cyanocobalamin (I) or hydroxocobalamin (II).

Finally, the use of a reaction solvent is not particularly limited, andin the case of using a solvent, it is not particularly limited as far asit is inert to cyanocobalamin (I), hydroxocobalamin (II),trimethylsulfur derivative (VI) or methylcobalamin (V). The reactionsolvent is usually an aqueous solution or a hydrous organic solvent. Asthe organic solvent, preferred is usually a water-soluble one, andexamples thereof include lower alcohols such as methanol, ethanol,propanol, isopropanol, butanol, isobutanol, sec-butanol and t-butanol;various esters such as methyl formate, ethyl formate, methyl acetate,ethyl acetate and isopropyl acetate; various ketones such as acetone,2-butanone and 3-methyl-2-butanone; cyclic ethers such as THF anddioxane; acetonitrile, DMF, DMSO, pyridine etc.; and mixtures containingone or more of them.

The reaction temperature in the present invention is also notparticularly limited, but the reaction is conducted at a temperature ofusually 0 to 90° C., preferably 10 to 70° C. and more preferably 15 to50° C.

A more preferred result is obtained by conducting the reaction under astream of an inert gas such as nitrogen and/or in the dark place (underinfrared ray).

In order to explain the present invention specifically, Examples will bedescribed in the following, but the invention is by no means limitedthereto.

EXAMPLES Example 1 Synthesis of Methylcobalamin

The present Example was conducted in the dark place (under infraredray).

To 260 ml of ion-exchanged water were added 20 g of cyanocobalamin, 6.02g of trimethylsulfonium iodide and 800 mg of iron(II) sulfateheptahydrate. The mixture was heated in a water bath and, afterreplacing the atmosphere of the system by nitrogen, a solution of sodiumborohydride (8 g)/2N sodium hydroxide (0.2 ml)/water (40 ml) and 15 mlof 2-butanone were added dropwise thereto under stirring at an innertemperature of 40° C. over 20 minutes, respectively. After stirring for15 minutes as it was, the mixture was cooled to 15° C. Further, 15 ml of2-butanone was added thereto, followed by stirring overnight. Theprecipitates were collected by filtration and dried, to give 21.4 g of acrude product of the title compound. Thereto was added 200 ml of a 50%acetone aqueous solution, and the mixture was heated, adjusted to pH 6.5with concentrated hydrochloric acid and then filtered. After washingwith 40 ml of a 50% acetone aqueous solution, 630 ml of acetone wasadded dropwise to the filtrate, followed by stirring at 15° C.overnight. Precipitated crystals were collected by filtration and dried,to give 17 g of the title compound (yield 86%).

Physical Properties of Mecobalamin Obtained

The hydrochloride buffer (pH 2.0): UV_(max) was detected at 264-266,303-307 and 459-462 nm.

The phosphate buffer (pH 7.0): UV_(max) was detected at 266-269, 341-344and 520-524 nm.

Referential values of UV_(max) (Merck Index, 12th edition)

Example 2 Synthesis of Mecobalamin

The present example was conducted in the dark place (under infraredray).

To 1.3 l of ion-exchanged water were added 100 g of cyanocobalamin and32.46 g of trimethylsulfoxonium iodide. After replacing the atmosphereof the system by nitrogen, the mixture was heated in a water bath and asolution of sodium borohydride (40 g)/2N sodium hydroxide (2 ml)/water(200 ml) was added dropwise thereto under stirring at an innertemperature of 40° C. over 30 minutes. After stirring for 1 hour as itwas, the mixture was cooled to room temperature and then stirredovernight. The precipitates were collected by filtration and dried, togive a crude product of the title compound. Thereto was added 1 l of a50% acetone aqueous solution, and the mixture was heated, adjusted to pH6.5 with concentrated hydrochloric acid and then filtered. After washingwith 400 ml of a 50% acetone aqueous solution, 2.8 l of acetone wasadded dropwise thereto and the mixture was stirred at 17° C. overnight.Precipitated crystals were collected by filtration and dried, to give 90g of the title compound (yield 91%).

Example 3 Synthesis of Mecobalamin

The present Example was conducted in the dark place (under infraredray).

To 1.3 l of ion-exchanged water were added 100 g of cyanocobalamin and32.46 g of trimethylsulfoxonium iodide, 4 g of iron(II) sulfateheptahydrate and 100 ml of 2-butanone. Under a nitrogen stream, themixture was heated in a water bath and a solution of sodium borohydride(40 g)/2N sodium hydroxide (1 ml)/water (200 ml) was added dropwisethereto under stirring at an inner temperature of 40° C. over 30minutes. After stirring for 30 minutes as it was, the mixture wasreturned to room temperature and further stirred overnight. Theprecipitates were collected by filtration and dried, to give 123 g of acrude product of the title compound. Thereto was added 1 l of a 50%acetone aqueous solution, and the mixture was heated at 35° C., adjustedto pH 7.0 with concentrated hydrochloric acid and then filtered. 2.8 lof acetone was added dropwise thereto, followed by stirring overnight.Precipitated crystals were collected by filtration and dried, to give93.2 g of the title compound (yield 94%).

Example 4 Synthesis of Mecobalamin

The present example was conducted in the dark place (under infraredray).

To 390 ml of ion-exchanged water were added 30 g of cyanocobalamin,14.61 g of trimethylsulfoxonium iodide, 900 mg of iron (II) sulfateheptahydrate, 900 mg of cobalt chloride hexahydrate and 22.5 ml of2-butanone. After replacing the atmosphere of the system by nitrogen,the mixture was heated in a water bath and a solution of sodiumborohydride (12 g)/2N sodium hydroxide (1 ml)/water (60 ml) was addeddropwise thereto under stirring at an inner temperature of 20° C. Afterstirring for 3 hours as it was, the mixture was cooled to 10° C. andthen stirred overnight. Thereto was added 24 ml of 3-methyl-2-butanone,followed by stirring for 2 hours. Then, the precipitates were collectedby filtration and dried, to give 35 g of a crude product of the titlecompound. Thereto was added 300 ml of a 50% methanol aqueous solutionand the mixture was heated at 35° C, filtered and washed with 120 ml ofa 50% methanol aqueous solution. After the mixture was adjusted to pH7.0 with concentrated hydrochloric acid, 1365 ml of acetone was addeddropwise thereto and the mixture was stirred at 10° C. overnight.Precipitated crystals were collected by filtration and dried, to give25.9 g of the title compound (yield 86.3%).

Example 5 Synthesis of Mecobalamin

The present example was conducted in the dark place (under infraredray).

To 130 ml of ion-exchanged water were added 10 g of cyanocobalamin, 3.83g of trimethylsulfoxonium bromide, 700 mg of iron (II) sulfateheptahydrate and 7.5 ml of 2-butanone. After replacing the atmosphere ofthe system by nitrogen, the mixture was heated in a water bath and asolution of sodium borohydride (4 g)/2N sodium hydroxide (0.2 ml)/water(20 ml) was added dropwise thereto under stirring at an innertemperature of 35° C. After stirring for 3 hours as it was, the mixturewas cooled to 15° C. and then stirred overnight. Thereto was added 7.5ml of 2-butanone, followed by stirring for 2 hours. Then, theprecipitates were collected by filtration and dried, to give a crudeproduct of the title compound. Thereto was added 140 ml of a 50% acetoneaqueous solution, and the mixture was heated at 45° C., filtered andwashed with 60 ml of a 50% acetone aqueous solution. After the mixturewas adjusted to pH 6.5 with concentrated hydrochloric acid, 475 ml ofacetone was added dropwise thereto and the mixture was stirred at 20° C.overnight. Precipitated crystals were collected by filtration and dried,to give 8.86 g of the title compound (yield 89.3%).

Example 6 Synthesis of Mecobalamin

The present example was conducted in the dark place (under infraredray).

To 650 ml of ion-exchanged water were added 50 g of cyanocobalamin,19.51 g of trimethylsulfoxonium bromide, 3.5 g of cobalt chloridehexahydrate and 37.5 ml of 2-butanone. After replacing the atmosphere ofthe system by nitrogen, the mixture was heated in a water bath and asolution of sodium borohydride (20 g)/2N sodium hydroxide (1 ml)/water(100 ml) was added dropwise thereto under stirring at an innertemperature of 35° C. After stirring for 2 hours as it was, the mixturewas cooled to 15° C. and then stirred overnight. Thereto was added 37.5ml of 2-butanone, followed by stirring for 1 hour. Then, theprecipitates were collected by filtration and dried, to give a crudeproduct of the title compound. Thereto was added 700 ml of a 50%methanol aqueous solution, and the mixture was heated at 40° C.,filtered and washed with 300 ml of a 50% acetone aqueous solution. Afterthe mixture was adjusted to pH 6.5 with concentrated hydrochloric acid,methanol was evaporated. To the residue was added dropwise 2250 ml ofacetone, followed by stirring at 20° C. overnight. Precipitated crystalswere collected by filtration and dried, to give 45.0 g of the titlecompound (yield 90.7%).

Example 7 Synthesis of Mecobalamin

The present example was conducted in the dark place (under infraredray).

To 130 ml of ion-exchanged water were added 10 g of cyanocobalamin, 3.48g of trimethylsulfonium bromide, 700 mg of cobalt chloride hexahydrate,and 7.5 ml of 2-butanone. After replacing the atmosphere of the systemby nitrogen, the whole was warmed on a water bath and a solution ofsodium borohydride (4 g)/2N sodium hydroxide (0.2 ml)/water (20 ml) wasadded dropwise thereto under stirring at an inner temperature of 35° C.After stirring for 3 hours as it was, the mixture was cooled to 15° C.and then stirred overnight. Thereto was added 7.5 ml of butanone,followed by stirring for 2 hours. Then, the precipitates were collectedby filtration and dried, to give a crude product of the title compound.Thereto was added 140 ml of a 50% acetone aqueous solution and themixture was heated at 45° C., filtered and washed with 60 ml of a 50%acetone aqueous solution. After the mixture was adjusted to pH 6.5 withconcentrated hydrochloric acid, 475 ml of acetone was added dropwisethereto and the mixture was stirred at 20° C. overnight. Precipitatedcrystals were collected by filtration and dried, to give 8.94 g of thetitle compound (yield 90.1%).

Example 8 Synthesis of Mecobalamin

The present Example was conducted in the dark place (under infraredray).

To 130 ml of ion-exchanged water were added 10 g of cyanocobalamin, 2.85g of trimethylsulfoxonium chloride, 700 mg of iron (II) sulfateheptahydrate and 7.5 ml of 2-butanone. After replacing the atmosphere ofthe system by nitrogen, the mixture was heated in a water bath. Asolution of sodium borohydride (4 g)/2N sodium hydroxide (0.5 ml)/water(20 ml) was added dropwise thereto under stirring at an innertemperature of 35° C. After stirring for 3 hours as it was, the mixturewas cooled to 15° C. and then stirred overnight. Thereto was added 7.5ml of butanone, followed by stirring for 2 hours. Then, the precipitateswere collected by filtration and dried, to give 35 g of a crude productof the title compound. Thereto was added 140 ml of a 50% acetone aqueoussolution. The mixture was heated at 45° C., filtered and washed with 60ml of a 50% acetone aqueous solution. After the mixture was adjusted topH 6.5 with concentrated hydrochloric acid, 475 ml of acetone was addeddropwise thereto and the mixture was stirred at 20° C. overnight.Precipitated crystals were collected by filtration and dried, to give8.92 g of the title compound (yield 89.9%).

What is claimed is:
 1. A process for producing methylcobalamin (V),which comprises the step of methylating cyanocobalamin (I) orhydroxocobalamin (II) represented by the following formula:

R²=CN: Cyanocobalamin (I) R²=OH: Hydroxocobalamin (II) R²=CH₃:Methylcobalamin (V) in the presence of a reducing agent (III) and awater-soluble methylating agent (IV) wherein the water-solublemethylating agent is represented by the formula (VI)

and wherein X means a halogen atom or methoxysulfonyloxy group; and nmeans 0 or
 1. 2. A process for producing methylcobalamin (V), whichcomprises the step of methylating cyanocobalamin (I) or hydroxocobalamin(II) in the presence of a reducing agent (III) and a water-solublemethylating agent (IV) in an aqueous solution or a hydrous organicsolvent; wherein the water-soluble methylating agent is represented bythe formula (VI)

and wherein X means a halogen atom or methoxysulfonyloxy group; and nmeans 0 or
 1. 3. A process for producing methylcobalamin (V), whichcomprises the steps of methylating cyanocobalamin (I) orhydroxocobalamin (II) in the presence of a reducing agent (III) and awater-soluble methylating agent (IV) in an aqueous solution or a hydrousorganic solvent; wherein the water-soluble methylating agent isrepresented by the formula (VI)

and wherein X means a halogen atom or methyoxysulfonyloxy group; andthen precipitating the reaction product as crystals or precipitates. 4.A process for producing methylcobalamin (V), which comprises the stepsof methylating cyanocobalamin (I) or hydroxocobalamin (II) in thepresence of a cyan ion-trapping agent, a reducing agent (III) and awater-soluble methylating agent (IV) in an aqueous solution or a hydrousorganic solvent wherein the water-soluble methylating agent isrepresented by the formula (VI)

and wherein X means a halogen atom or methoxysulfonyloxy group; and nmeans 0 or 1; and then precipitating the reaction product as crystals orprecipitates.
 5. The process for producing methylcobalamin (V) accordingto any one of claims 1 to 4, wherein the trimethylsulfur derivative (VI)is at least one selected from trimethylsulfoxonium iodide,trimethylsulfonium iodide, trimethylsulfoxonium bromide,trimethylsulfonium bromide, trimethylsulfoxonium chloride andtrimethylsulfonium chloride.
 6. The process for producingmethylcobalamin (V) according to any of claims 1 to 4, wherein thereducing agent (III) is sodium borohydride.
 7. The process for producingmethylcobalamin (V) according to claim 4, wherein the cyan ion-trappingagent is at least one selected from ferrous sulfate and cobalt chloride.8. The process for producing methylcobalamin (V) according to claim 4 or7, wherein the amount of the cyan ion-trapping agent is from 1 to 30% byweight relative to cyanocobalamin (I) or hydroxocobalamin (II).
 9. Theprocess for producing methylcobalamin (V) according to any of claims 1to 4, wherein the trimethylsulfur derivative (VI) is at least oneselected from trimethylsulfoxonium iodide, trimethylsulfonium iodide,trimethylsulfoxonium bromide, trimethylsulfonium bromide,trimethylsulfoxonium chloride and trimethylsulfonium chloride; and thereducing agent (III) is sodium borohydride.
 10. The process forproducing methylcobalamin (V) according to claim 4, wherein thetrimethylsulfur derivative (VI) is at least one selected fromtrimethylsulfoxonium iodide, trimethylsulfonium iodide,trimethylsulfoxonium bromide, trimethylsulfonium bromide,trimethylsulfoxonium chloride and trimethylsulfonium chloride; thereducing agent (III) is sodium borohydride; and the cyan ion-trappingagent is at least one selected from ferrous sulfate and cobalt chloride.